Angiogenesis treatment for ischemic diseases is intended to enhance neovascularization by delivering vascularization factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), or DNA vectors that encode these proteins, to ischemized tissue (Non-patent Documents 1 to 3). VEGF is produced in the initial stage of the angiogenesis cascade and involved in the initial activation of endothelial cells, hence said to be an important factor of vascular development. It has been reported that in the skin of VEGF-transgenic mice, a very large number of blood vessels exhibiting excess permeabiity are formed (Non-patent Document 4). FGF2 has been reported to act as a mitogen on both endothelial cells and wall cells, and the role thereof in angiogenesis has also been identified (Non-patent Documents 3 and 5). As stated above, a very large number of studies have been conducted on vascularization therapies using VEGF or FGF2 alone.
In clinical studies, the safety of VEGF and FGF2 in delivery is demonstrated at the stage of phase I, but no expected efficacy has been demonstrated in phase II (Non-patent Documents 6 to 8). As a result, it has been suggested that to induce functional blood vessels, it is unsatisfactory to administer a single vascularization factor alone.
Later, research into vascularization therapy became focused on administering vascularization factors in combination. Specifically, a combination of VEGF and FGF2, or a combination of VEGF or FGF2 and another vascularization factor like angiopoietin 1 (Ang-1) or platelet-derived growth factor-BB (PDGF-BB), has been reported to have synergistic action effective in neovascularization in in vitro and in vivo experiments (Non-patent Documents 9 to 13). These results demonstrate the complexity of the mechanism for vascularization involving the temporarily and spatially integrated expression of a large number of vascularization factors. Constructing functional blood vessels requires three complex processes, i.e., vasculogenesis, angiogenesis, and arteriogenesis; however, because no vascularization factors that act on all these processes in common are currently available, a single vascularization factor alone is inadequately effective, and it is thought that a satisfactory effect is difficult to obtain even when several kinds of vascularization factors are combined. Furthermore, in current clinical studies using a gene of vascularization factor and the like, methods of administration by intramuscular injection are employed; however, because intramuscular injection is highly invasive to tissue, and also because its gene transfer effect is regionally limited so that the transfer efficiency is not always satisfactory, it seems to be necessary to improve the method of administering a vascularization factor.
While there is a need for discovering a radical factor that acts on all angiogenesis processes and developing countermeasures, what can become a key thereto is hypoxia-inducible factor (HIF). Known members of the HIF family include HIF-1, HIF-2, HIF-3 and the like. HIF-1 is a hetero-dimer consisting of an α subunit and a β subunit, functioning as a pivotal regulatory factor for oxygen homeostasis. It is known that the production of vascularization factors such as VEGF, FGF2, and Ang-1 is induced directly or indirectly by the α subunit of HIF-1 (HIF-1α). In the presence of oxygen, however, HIF-1α is hydroxylated by prolyl hydroxylase domain-2 (PHD2); the hydroxylated HIF-1α is then decomposed by E3 ubiquitin ligase complex. In a study using PHD2-hetero-deficient mice, it was shown that tumor metastasis is suppressed via normalization of vascular endothelium, suggesting that inhibiting PHD2 leads to cancer treatment (Non-patent. Document 14).
The present inventors attempted to introduce a PHD2-siRNA expression plasmid into mouse fibroblasts to achieve silencing of the PHD2 gene, and reported that the expression of VEGF and FGF2 was significantly induced, and that angiogenesis was induced when the introduced cells were subcutaneously transplanted to mice (Non-patent Document 15).
Meanwhile, the present inventors reported that a polyion complex of a nucleic acid and a block copolymer is useful as a delivery system for nucleic acids such as DNA (Patent Documents 1 to 5). However, no delivery system for nucleic acids is known to be effective in the treatment of ischemic diseases.